Apparatus and method for synthesizing

ABSTRACT

An apparatus and method for synthesizing a combinatorial library comprising a plurality of chemical compounds such that the chemical composition of each compound is easily tracked. The library compounds are synthesized on solid-phase supports, which are spatially arranged in frames during synthesis according to a predetermined protocol, such that each solid-phase support passes through a series of unique spatial 2D or 3D addresses by which the chemical composition of each compound may be determined at any point during synthesis. Solid-phase supports include hollow tubular-shaped lanterns and gears.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. patent application Ser.No. 09/449,222, filed Nov. 24, 1999, which is a continuation-in-part ofU.S. patent application Ser. No. 09/082,038, filed May 20, 1998, theentire disclosures of which are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to the field of combinatoriallibraries. More specifically, the invention relates to methods ofsynthesis utilizing arrays of solid-phase supports to produce acombinatorial library of chemical compounds and, additionally, theapparatuses used to carry out those methods.

BACKGROUND OF THE INVENTION

[0003] Citation or identification of any reference in section 2 or anysection of this application shall not be construed as an admission thatsuch reference is available as prior art to the present invention.

[0004] A combinatorial library is a collection of multiple species ofchemical compounds comprised of smaller subunits or monomers.Combinatorial libraries come in a variety of sizes, ranging from a fewhundred to several thousand species of chemical compounds. There arealso a variety of library types, including oligomeric and polymericlibraries comprised of compounds such as peptides, carbohydrates,oligonucleotides, and small organic molecules, etc. Such libraries havea variety of uses, such as identifying and organic molecules, etc. Suchlibraries have a variety of uses, such as identifying and characterizingligands capable of binding an acceptor molecule or mediating abiological activity of interest.

[0005] The library compounds may comprise any type of molecule of anytype of subunits or monomers, including polymers wherein the monomersare chemically connected by any sort of chemical bond such as covalent,ionic, coordination, chelation bonding, etc., which those skilled in theart will recognize can be synthesized on a solid-phase support. The termpolymer as used herein includes those compounds conventionally calledheteropolymers, i.e., arbitrarily large molecules composed of varyingmonomers, wherein the monomers are linked by means of a repeatingchemical bond or structure. The polymers of the invention of this typesare composed of subunits or monomers that can include any bi-functionalorganic or herteronuclear molecule including, but not limited to aminoacids, amino hydroxyls, amino isocyanates, diamines, hydroxycarboxylicacids, oxycarbonylcarboxylic acids, aminoaldehydes, nitroamines,thioalkyls, and haloalkyls. In the disclosure of the present invention,the terms “monomer,” “subunits” and “building blocks” will be usedinterchangeably to mean any type of chemical building block of moleculethat may be formed upon a solid-phase support.

[0006] Various techniques for synthesizing libraries of compounds onsolid-phase supports are known in the art. Solid-phase supports aretypically polymeric objects with surfaces that are functionalized tobind with subunits or monomers to form the compounds of the library.Synthesis of one library typically involves a large number ofsolid-phase supports. Solid-phase supports known in the art include,among others, polystyrene resin beads, cotton threads, and membranesheets of polytetrafluoroethylene (“PTFE”).

[0007] To make a combinatorial library, solid-phase supports are reactedwith a one or more subunits of the compounds and with one or morenumbers of reagents in a carefully controlled, predetermined sequence ofchemical reactions. In other words, the library subunits are “grown” onthe solid-phase supports. The larger the library, the greater the numberof reactions required, complicating the task of keeping track of thechemical composition of the multiple species of compounds that make upthe library. Thus, it is important to have methods and apparatuses whichfacilitate the efficient production of large numbers of chemicalcompounds, yet allow convenient tracking of the compounds over a numberof reaction steps necessary to make the compounds.

[0008] One method of making combinatorial libraries is described in U.S.Pat. No. 5,510,240 to Lam et al. (“Lam '240 patent”), the disclosure ofwhich is incorporated herein by reference in its entirety. Morespecifically, the Lam '240 patent discloses a split and mix method ofsynthesizing combinatorial libraries of bio-oligomers on resin beads, incertain embodiments of which the library contains all possiblecombinations of monomer subunits of which the bio-oligomers arecomposed. Although there may be several resin beads containing the samespecies of bio-oligomer, each resin bead contains only one species ofbio-oligomer.

[0009] Another example of a method of making combinatorial libraries ondivisible solid-phase supports is described in U.S. Pat. No. 5,688,696to Lebl (“Lebl '696 patent”), the disclosure of which is incorporatedherein by reference in its entirety. In the method disclosed in the Lebl'696, each of a set of predetermined species of test compounds ispresent on a predetermined number of solid-phase supports—preferably ononly one—and each solid-phase support has only a single species of testcompound.

[0010] The use of radio-frequency identification (“RFID”) chips torecord the steps of library synthesis is also known. See, for example,U.S. Pat. Nos. 5,741,462, 5,770,455, and 5,751,629, as well as WO98/15826.

[0011] A method and apparatus for synthesis of a combinatorial libraryusing a 3-D array of reaction zones is provided in Glaxo's WO 99/32219(“Glaxo Application”). This application discloses stackable frameshaving a plurality of holes. Membranes, which act as the solid supports,are trapped between stacked frames, and these membranes are exposed atthe frame holes. In an alternative embodiment, solid support beads areplaced on flow-through sieves that allow flow-through of reagents aroundthe support beads. Reagents are pumped in from the top and vacated atthe bottom or, alternatively, pumped in from the bottom and vacated atthe top. The apparatus disclosed allows reagents to be delivered togroups of supports in the X-Z planes or in the Y-Z planes duringsynthesis steps.

[0012] The Glaxo Application also employs a 3-D (X-Y-Z) array ofsupports. However, instead of using a containment apparatus having truewells in which solid supports are stacked, the Glaxo method employsstackable 2-D (X-Y) frames. The Glaxo Application discloses two distinctembodiments of stackable frame structures. One embodiment sandwiches amembrane between stacked frames, the frames having a plurality of holes.The membranes are solid-phase supports which are held between theframes. The frame holes expose the membranes. The membranes also haveholes to allow reagents to pass through the layers of membranes andcontact other membranes in the vertical “column” of the array. Anotherembodiment has sieves in place of the membranes, and free solid supportsare placed on each sieve between the frames. The sieves allow reagentsto flow vertically from top to the bottom of the stacked 3-D arraycontacting a vertical column of solid-phase supports resting on sieves.

[0013] A major disadvantage with Glaxo's apparatus and method, however,is that after the synthesis is completed, the solid supports, whether asthe membrane or the solid-phase support beads suspended on the sieve,are not easily freed from the stacked array while retaining theirspatial identities. The frames must be taken apart one by one to gainaccess to the supports and to provide some means to retain theidentities of each support. This requires a burdensome additional stepthat makes the apparatuses disclosed less attractive for commercialproduction of libraries.

[0014] While methods exist in the art that can be used to produce alibrary of compounds, there is still a need for methods and apparatuseseffective for commercial use to build a large library of compoundsquickly and with a minimum of cost. Thus, there is still a need foralternative methods of synthesis that use 2-D or 3-D arrays ofsolid-phase support as part of the synthesis process for the purpose ofcommercially making large libraries of compounds efficiently.

[0015] Moreover, there is still a need for apparatuses and methods forefficiently synthesizing extremely large libraries, e.g., greater than100,000 compounds, using 2-D or 3-D arrays as tools in the synthesis.

SUMMARY OF THE INVENTION

[0016] The present invention provides methods and apparatuses that use2-D or 3-D array of solid-phase supports and that may be used tocommercially synthesize a library of compounds. In particular a methodis provided which may be commercially used to produce large librarieshaving between about 100,000 to 200,000 compounds. A number ofembodiments of methods and apparatuses for synthesizing libraries ofcompounds are provided herein in accordance with the present invention.

[0017] In a first embodiment, in accordance with the present invention,a 3-D array of solid-phase supports is used to provide parallelsynthesis. One embodiment of the apparatus which provides this 3-D arrayis a containment device which has a plurality of wells wherein discretesolid-phase supports can be placed into and stacked in a column. Inanother embodiment of the apparatus, a 3-D array is formed by stacking aplurality of 2-D frames which have solid-phase supports arranged in anorderly X-Y array. The frames have a plurality of holes arranged in anorderly X-Y array and solid-phase supports can be friction fitted orinterlocked into these holes to temporarily hold the supports to theframe during synthesis. Alternatively, the supports can be physicallyattached to the frames in a manner in which, when desired, they can beeasily cut from the frame. Associated with this 3-D array, specificembodiments of the apparatuses are disclosed, in accordance with thepresent invention, including a 3-D containment plate which hasdouble-drilled holes, a gear-shaped solid-phase supports (“gear”)designed to be friction fitted or interlocked into 2-D frame holes, alantern-shaped solid-phase supports (“lantern”), and ring supports usedin conjunction with a containment device having a plurality of wells.

[0018] A specific synthesis method is provided, which can be used withan apparatus having a 3-D arrangement of solid-phase supports, inaccordance with the present invention. A preferred method provides amonomer or subunit diversity to the library compounds on the solid-phasesupports between the X-Y layers in the Z direction. The methodcomprises: providing reagents to react with solid-phase supports in theX-Z layers, providing reagents to react with solid-phase supports in Y-Zlayers, and retrieving columns of solid-phase supports, while retainingtheir spatial relationships.

[0019] A defining characteristic of this first method embodiment using a3-D array of support is once the array is formed, the supports aregenerally not moved during the subsequent synthesis steps. Reagents forreacting with the supports are brought to the array and usually aparticular reagent is delivered only to a subset of the supports in the3D array. Additionally, the size of the library of compound will belimited by the size of the 3-D array.

[0020] In a second method embodiment, in accordance with the presentinvention, the same stackable frames are used as in the firstembodiment. Frames having X-Y arrays of solid-phase supports are stackedinto 3-D arrays (“stacks”). Instead of a single 3-D array, in thissecond embodiment a multiple N number of stacks are formed inpreparation for making a library of compounds.

[0021] In the first synthesis step, each stack numbered 1 to N iscompletely immersed into separate reactors 1 to N respectively, eachreactor having a distinct reagent and a subunit is attached to eachsupport in the stack. After each stack is removed from its reactor, afirst randomization occurs by taking one and only one layer (frame) ofeach original stack, combining these layers to form a new stack. Thus,the first layer or frame from each original stack is grouped to create afirst new stack, the second layer or frame from each original stack isgrouped to create a second new stack. This reshuffling process isrepeated until all the original frames of each old stack are transferredto a set of N number of new stacks. Then, in the second synthesis step,each new stack from 1 to N is immersed in a set of reactors, eachreactor having a different reagent.

[0022] In the second randomization step, one vertical column ofsolid-phase supports is removed from each new stack keeping the spatialidentification of the supports intact and then reassembled to make a newgrouping of 3-D supports. Another vertical column of supports is removedfrom each new stack and regrouped to another grouping of 3-D supports.This process is repeated until all the supports in the new stack arrayshave been regrouped into a N number of new 3-D arrays. In thisregrouping, randomization step, only one vertical column of supports istaken from each new stack to make a new grouping of supports. In thethird and final synthesis step, the new groupings of supports are eachput into separate 1 to N reactors, each reactor having a differentreagent.

[0023] The apparatuses used with this second embodiment are the same asused in the first embodiment. A preferred embodiment of the frame andsolid-phase support is a 2-D frame having a plurality of holes arrangedin an X-Y rectangular order. A preferred apparatus comprises gears orlanterns friction fitted or interlocked into the plurality of holes.Additionally, reactors having a capacity large enough for immersion of3-D stacks are needed.

[0024] The defining characteristics of this second embodiment are: (a)many N number of 3-D original stacks are formed; (b) the original stacksdo not have solid supports which have a subunit attached in contrast toembodiment one; (c) the solid supports are disturbed from the original3-D arrays because the supports are moved during the synthesis processwhen the frames are reshuffled and vertical columns of supports areregrouped; and (d) every solid support in each 3-D stack is completelyimmersed in the reagent during a synthesis step because the stack isbrought to the reagents/reactors. The second embodiment lends itself tolarge scale production of libraries of compounds because the finalnumber of unique compounds is based on the number N of original stacksmade.

[0025] The third embodiment, in accordance with the present invention,uses 2-D frames in a “sort and combine” method of synthesis. There is nostacking of the frames into a 3-D array. Instead, the 2-D frames aresplit during synthesis of the combinatorial library. The method of thisthird embodiment can be implemented by automation since no rods arerequired and may be used to generate large libraries, having betweenabout 100,000 to 200,000 compounds.

[0026] In this method, a Q number of 2-D frames is chosen. The 2-Dframes have rows and columns. Solid supports are placed into reagentsfor a first synthesis step. Solid supports thus reacted with a singlesubunit are placed into the frame holes such that the frame has columnsof supports which have the same subunit, but between columns, there is adiversity of subunits. This placement provides the first randomization.Each Q number of frames is initially identically prepared. Next, in asecond synthesis step, the Q frames are placed into 1 to Q reactors,each having a different reagent. After removal from the reactors, the Qnumber of frames are split up into subframes to provide the secondrandomization. M new groups of subframes are regrouped by taking one andonly one subframe from each original frame. M represents the number ofsubframes a frame has been split into. The M new groups of subframes,each are immersed into 1 to M reactors, each reactor having a differentreagent. After final synthesis the supports are detached from thesubframes and placed into a labeled cleavage plate.

[0027] The total number of unique compounds in the library is Q×M×N,where N is the number of columns present in the original 2-D frames, andQ is arbitrarily chosen. The size of the library will be controlled bychoice of three variables Q, M and N.

[0028] The preferred apparatuses used in this embodiment are 2-D frames.Solid-phase supports such as gears are friction fitted or interlockedinto the plurality of holes in the frame. The additional feature of theframe is that it must be easily splittable into subframes. Reactors areneed which have capacity for accepting groups of subframes.Additionally, in accordance with a preferred embodiment of the presentinvention, a 2 row subframe having a RIFD chip is disclosed.

[0029] The defining characteristics of this third method embodiment are:(a) user choice of the number of frames Q to use in the synthesis; (b)the solid supports are disturbed from the original 2-D arrays becausethe supports are moved during the synthesis process when the frames aresplit and regrouped; and (c) every solid support in each 2-D frame or2-D subframe is completely immersed in the reagent during a synthesisstep because the frame or group of frames is brought to thereagents/reactors. The third embodiment lends itself to large scaleproduction of libraries of compounds because the final number of uniquecompounds is based on the number Q of original frames used.

[0030] All three method embodiments use 2-D or 3-D arrays of supportsheld in frames to facilitate parallel synthesis on solid-phase supportsand to provide spatial identification and thus the synthesis history ofthe compound produced on a particular support.

[0031] There is interchangeability of apparatuses used in the variousembodiments described above, in accordance with the present invention.For example, the supports, frames, rods and devices for removing thesupports from the frames are interchangeable. A gear design of solidsupport for use with 2-D frames is provided in accordance with thepresent invention. A new embodiment of a 3-D containment plate havingdouble-drilled holes and RFID chip is provided in accordance with thepresent invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Reference is next made to a brief description of the drawings,which are intended to illustrate a number of embodiments of theapparatus and method of making a combinatorial library according to thepresent invention. The drawings and detailed descriptions which followare intended to be merely illustrative, and are not intended to limitthe scope of the invention as set forth in the appended claims.

[0033]FIG. 1a illustrates six flasks 20 a-20 f, having six differentreagents, and 96 solid supports used in the first embodiment of thepresent invention;

[0034]FIG. 1b provides a cutaway side view of a containment well showingthe first layer of solid-phase supports after being distributed from thefirst flask;

[0035]FIG. 1c shows a top view of the 96 well plate;

[0036]FIG. 1d shows another cutaway side view of the containmentapparatus with wells containing all six layers of solid-phase supports,wherein each layer has a different subunit or building block;

[0037]FIG. 2 is a combined diagram and top view of the 96 wellcontainment apparatus, wherein R2(A) through R2(H) represent differentreagents delivered into the rows of wells (X direction of the array) ofthe apparatus in the second synthesis step;

[0038]FIG. 3 is a combined diagram and top view of the 96 wellcontainment apparatus, wherein R3(A) through R3(L) represent differentreagents delivered into the columns of wells (Y direction of the array)of the apparatus in the third and final synthesis step;

[0039]FIG. 4 is a mixed side view and perspective view illustrating thefinal distribution of the solid-phase supports, in which each solidsupport now provides a unique compound and each layer is distributedinto one-layer cleavage plates, 38 a-f;

[0040]FIG. 5 is a perspective view of a single frame, showing thefriction fitted or interlocked gears in a plurality of holes, a stack offrames providing a 3-D array, and a complete set of original “stacks”consisting of 24 total stacks;

[0041]FIG. 6 is a perspective view illustrating the first randomizationstep involving taking one layer from each original stack to create a newstack and a set of new stacks;

[0042]FIG. 7 is a perspective view illustrating the second randomizationstep of the present method, wherein a single column of supports is takenfrom each new stack and regrouped to form a new group of supports in a3-D array and each column is then treated with reagents in a thirdrandomization step;

[0043]FIG. 8 is a top view of a gear-shaped solid support (“gear”) madeby Chiron;

[0044]FIG. 9 is a top view of a gear frame that may be used in allembodiments of the present invention;

[0045]FIG. 10 is a perspective view of a solid-phase support shaped as aring;

[0046]FIG. 11 is a top view of a 3-D containment plate havingdouble-drilled holes for wells;

[0047]FIG. 12 is a schematic diagram illustrating an embodiment of thepresent invention used to synthesize a library having 27 subunits;

[0048]FIG. 13 is a perspective view of a Chiron lantern solid support;

[0049]FIG. 14a is a perspective view of a frame used to containsolid-phase supports, the frame having an RFID chip; and

[0050]FIG. 14b is a top view of the frame shown in FIG. 14a.

DETAILED DESCRIPTION OF THE INVENTION

[0051] A detailed explanation of the methods and apparatuses inaccordance with the present invention with reference to the drawings isprovided as follows:

[0052] A. Synthesis of Compounds Using Frames Stacked to Provide a 3-DArray

[0053] One method of synthesizing solid supports was disclosed inco-pending Baum et. al. U.S. patent application Ser. No. 09/082,038 thedisclosure of which is incorporated by reference in its entirety. (“BaumApplication”) (See Baum Application, p. 6, para. 1 and p. 10, para. 2,for discussion of the method utilizing a 3-D array.) The method uses acontainment apparatus having a plurality of vertical wells. Freesolid-phase supports are “stacked” into each well, each supportphysically contacting adjacent supports within a well. (See BaumApplication, pp. 4-6, in particular, top of p. 5, lines 4-5, discussinga “plurality of discrete supports arranged in a plurality of columns inone or more wells.” See also FIGS. 12-17, 23-29 which describe variousembodiments of 3-D apparatuses containing a plurality of wells, whereinsupports are stacked, and pp. 6-7 which provide Figure captions anddiscussions of those Figures in pp. 8-41.) The stacking of supports inthe containment structure thereby provides an overall 3-D spatialarrangement of supports within the containment apparatus. Thus, afterstacking, each support in the 3-D array may be identified by its X-Y-Zposition in the array. Once the supports are placed inside the wells,reagents may be directed into sets of wells to react with the supportsduring steps of the synthesis process. Importantly, because of the openwell structure of the containment apparatus, when the synthesis stepsare completed, the supports can be easily retrieved from the wells,while retaining the spatial identification of each support.

[0054] An example implementing the specific steps of the method isillustrated FIG. 1. The solid support 10 must be of a type which is freeand can be stacked vertically in the containment wells. The solidsupport may be of various types including, but not limited to, thosedisclosed in co-pending U.S. application Ser. No. 09/082,038, as well asthose known in the art. (See Baum Application, FIGS. 12-17, 25-28, whichdescribe various embodiments of solid supports contained in wellswherein “discrete” or free supports are stacked; pp. 6-7 which provideFigure captions and discussion of those Figures in pp. 8-41 and ; p. 21at para. 1-2 discussing possible shapes of supports.) The solid supportsused in the following example is a commercially available Chironlantern, as shown in FIG. 13.

[0055] As shown in FIG. 1a, there are six flasks, 20 a-f, eachcontaining a different reagent, R1. As a first synthesis step 96lanterns 10 are placed into each flask to provide a total of 576lanterns reacted. The reagents in the flasks attaches to thefunctionalized surface of the lanterns, thereby forming first synthesisintermediates. It can be seen that six different types of synthesisintermediates are formed by placement in the six flasks, havingdifferent reagents.

[0056] Next, the 96 lanterns are taken from the first flask 20 a anddistributed into the 96 vertical wells 45 as the first X-Y layer (Z=1)of supports in the containment apparatus 35, as shown in FIG. 1b. Thelanterns from the next flask 20 b are then distributed in the samemanner forming the second layer of supports within the containmentapparatus. This process is repeated until all lanterns from eachremaining flask 20 c-f are distributed by layers into the containmentapparatus.

[0057] The containment apparatus 35 must be made of a material which isinert to reagents and can provide proper structural rigidity. A standard96-well plate, each well approximately 2 ml deep, can be used as 3-Dcontainment apparatus with the proper choice of stackable solidsupports. As provided in FIG. 1c, which shows a top view of thecontainment apparatus, the apparatus has 96 total wells placed in aneight by twelve arrangement in the X-Y plane. In the Z verticaldirection of the array, the well must have a depth to accommodate thetotal number of different reagents as shown in FIG. 1d. Because thereare six flasks, having six different reagents in the example depicted,the depth of the well must accommodate at least six lanterns.Ultimately, there will be a vertical stack of six lanterns in each well,and each ring will have attached a different subunit, monomer orbuilding block. The configuration or construction of any stackable solidsupport 10, including the example lanterns, should be designed with thedimensions to prevent relative movement of the supports within the wells45.

[0058] With the supports placed in 3-D array arrangement, the secondsynthesis step takes place. As shown in FIG. 2, reagent R2(A) isdirected to the first row of wells in the X direction of the array, therow consisting of 12 wells. The reagents bond to the particular monomerof each lantern to create second synthesis intermediates. Continuing theprocess, a different reagent R2(B) through R2(H), as shown in FIG. 3, isdirected to successive rows of the containment apparatus. At theconclusion of this step, 48 distinct compounds are formed in the array.

[0059] The third synthesis step repeats the previous steps by taking adifferent set of reagents, R3(A) to R3(L), and directing the reagentssuccessively into row groupings of eight wells pointed in the Ydirection of the array. The reagents react with the second synthesisintermediates to create the third and final synthesis product. At theconclusion of this step, there are a total of 576 distinct compoundsrepresenting each element of the X-Y-Z combinatorial array.

[0060] The last step, as shown in FIG. 4, is the transfer of the solidsupports (lanterns) within the containment apparatus 35 into to sixseparate 96-well plates 38 a-f. Each plate will accept only a single X-Ylayer of the original 3-D array of supports. Thus, the top X-Y layer ofsupports is transferred to plate 38 a. The next underlying X-Y layer ofsupports is transferred to plate 39 b. The process is repeated until alllayers have been transferred. The transfer should be performed in amanner which retains the spatial relationships of the supports. The newplates 38 a-f must be properly labeled to identify which X-Y layer iscontained, and thus, each transferred lantern may be identified by itsoriginal location in the X-Y-Z array of supports.

[0061] After the lanterns are transferred to single-layer 96 wellplates, the compounds still attached to the lanterns may be storedwithin these plates. Alternatively, the compounds may be cleaved fromthe lanterns using a cleavage solution. After cleavage, the compoundsmay be extracted onto another plate, dried and prepared for biologicalscreening or other purposes for which they may be suited in a mannerknown in the art.

[0062] In sum the synthesis system comprises: (a) a 3-D array ofsupports; (b) free solid supports; (c) a containment apparatus with aplurality of open wells in X-Y arrangement; and (d) means for removingvertical Z column array of supports from the well from the top or bottomof the 3-D array, once the rounds of synthesis are completed.

[0063] The synthesis method comprises: (a) providing free solidsupports; (b) providing a containment apparatus having a plurality ofopen wells; (c) stacking free solid supports into the wells to create a3-D array of supports; (d) delivering reagents to portions of the 3-Darray and; and (e) removing the supports in vertical Z columns.

[0064] A particular method of synthesis using the system abovecomprises: (a) providing an X-Y layer of supports all having onebuilding block attached and diversity of building blocks between X-Ylayers in the Z vertical direction of the 3-D array; (b) providingrandomization and synthesis by providing reagents first in the X-Zlayers and then Y-Z layers of the 3-D array; and (c) removing verticalcolumns of supports all at once through the well opening, therebypreserving the spatial information of the supports.

[0065] In accordance with one embodiment of the present invention, avariation of the above described method of combinatorial synthesis usinga single, 3-D array of supports is provided. The formation of the 3-Darray of supports is different in this embodiment. In contrast tostacking free solid supports into a separate well containment apparatus,in this variation frames of supports are stacked together to provide a3-D array of supports. Each 2-D frame defines a single X-Y layer ofsupports. When stacked, the frames form their own solid support reagentcontainment compartments, and therefore a separate containment apparatusis not needed.

[0066] The supports are either attached temporarily by some mechanicalmeans, such as friction fitting or interlocking into holes of theframes, or the supports come physically attached to the frames but in amanner whereby the supports may be easily cut from the frame.

[0067] Referring to FIG. 5, an example of a single, 3-D stacked frame 76is shown at frames having gears for solid-phase supports fitted intoholes by friction fit or interlocking. Only a single 3-D stack 76 isused in this synthesis embodiment. The method of synthesis is nearlyidentical to the process used with the open well 3-D containmentapparatus. In the first synthesis step, all supports in a single layeror frame are reacted with one type of reagent creating layer diversity.There are several ways to have a frame having all supports attach asingle building block. Assuming that free solid supports are used withframes with holes, in which the supports are friction fitted orinterlocked, a first way is to have free solid supports such as alantern or a gear reacted in a reactor such as a flask. The solidsupports are then inserted into the holes in a frame and held in placeby friction fit or some other means. A second way is to insert the solidsupports into the frame first, and immerse the entire frame in areactor. Assuming that the frame has integral supports attached,immersion of the entire frame into a reactor is the only alternative.Each frame must be immersed in its own reagent. Stacking the layers offrames thereby provides a diversity of monomers or building blocksbetween layers in the Z direction of the support array.

[0068] Once the frames have been stacked, the steps of synthesis andrandomization are identical as with the 3-D array using free solidsupports and a well containment apparatus. The only difference may be inthe last step of removing vertical columns of solid supports from thearray. If the solid supports are attached to the frame by friction fitor interlocking, a means must be used to remove individual columns ofsupports in the Z direction of the 3-D array. If lanterns are used asthe solid supports, a rod may be inserted through the holes of thelanterns to capture a single vertical column of rings. As shown in FIG.7 the rod 82 has a stop-end 90 on one end. The other end of the rod isinserted through the holes of the lanterns and then pulled to free thevertical column of lanterns from the stacked frames. The lanterns thuscaptured on the rod are spatially intact and may be labeled and stored.Additionally, each ring may be taken out and placed into a single layercleavage plate and further labeled. If the solid supports are integralto the frame, then there must be an intervening step of cutting thesupports from the frame with some cutting device.

[0069] Thus, the system comprises: (a) 3-D stackable frames; (b) meansfor temporarily attaching the supports to the 2-D frame; (c) means forremoving the solid supports without disassembly of the 3-D stack,retaining the 3-D spatial relationship of the solid supports; and (d) achannel means to allow reagents in a vertical column in the Z directionto allow supports to contact and react with the reagent directed intothe channel.

[0070] The method of synthesis is the same as described herein above.The only difference is the addition of an optional cutting step ifsupports are integral to the frames. A defining feature of this methodis that the reagent is brought to the stacked 3-D array. The finalcompounds formed are identified by their 3-D spatial locations.

[0071] B. Split-Mix Synthesis Using Stacked Frames and Rods

[0072] In accordance with another embodiment of the present invention, amethod is disclosed which uses multiple stacks of frames as shown inFIG. 5. The method involves (a) stacking of frames having a plurality ofsupports attached to the frames, forming a plurality of identicalstacked frames; (b) providing a first synthesis step comprisingimmersing each stack in a separate reactor to attach a building block toall of the solid supports in the stack of frames; (c) reshuffling theoriginal stacks, for example, such that each first layer of eachoriginal stack of frames is grouped in a new stack of frames, eachsecond layer of each original stack of frames is grouped in a new stackof frames, and this process is repeated until all the original stack offrames are reshuffled into new stack of frames; (d) providing a secondsynthesis step immersing these new stacks each into its own reactor toprovide the third step of synthesis; (e) reshuffling the stacks a secondtime by liberating the columns of supports from each 3-D stack, in amanner that retains the spatial relationship of the supports with theother supports of each column in the Z direction and groupingcorresponding columns of supports from the first re-shuffled stacks toform new final stacks; and (f) providing a third synthesis step byimmersing each new final stack into its own reactor.

[0073]FIG. 5 provides a specific example of the method using particularembodiments of the apparatus. Frames having 48 holes are shown. Thesolid supports depicted are shaped as gears which may be placed insidethe holes by friction fit or interlocking. As shown in the particularexample, a complex library having 27,648 compounds is synthesized onsolid-phase supports, wherein the compounds are ultimately arranged in a3-D array, and wherein each compound has a unique 3-D spatial address.In this example, the solid-phase supports comprise gears, which willdiscussed in more detail below.

[0074] As shown in FIG. 5, gear-shaped solid supports 70 (“gears”) areplaced in plastic gear frames 72. Each frame has a six-by-eightarrangement of holes, which holes have 48 gears inserted. The 24 totalframes 72 are stacked together to provide a 3-D stack 76. In thisexample, 24 identical 3-D set of stacks 74 are created. Given that thereare 24 total stacks, 24 frames in each stack, and 48 gears in eachframe, the total number of gears in the twenty-four stacks is 27,648.Each stack has 1152 gears.

[0075] After the total of 24 stacks are formed, each of these stacks isimmersed in its own reactor for the first round of synthesis. Becausethere are 24 stacks, there are 24 corresponding reactors, each reactorcontaining a unique subunit, monomer or building block to be attached tothe gears. After completion of the first synthesis, each of these 1152gears in a stack has attached a single building block.

[0076] After the first round of synthesis is completed, a firstrandomization step follows by reshuffling the 24 original stacks into anew stacks. As illustrated in FIG. 6, frames 72 in the first set of 24original frame stacks 74 are rearranged in a predetermined pattern intoa second set of frame stacks 78. In the second set of stacks 78, becauseall the frames must be accounted for, there are again 24 frame stacks,and each stack consists of 24 frames.

[0077] In a particular example of a predetermined pattern, as shown inFIG. 6, the top-most frame of each of the frame stacks in the originalframe stack set 74 is arranged in a new frame stack, identified by thelabel (r1, c1) depicted in newly reshuffled frame stack 78. Similarly,the second layer frames of original frame stacks 74 are arranged in anew frame stack, identified by the label (r1, c2). In this way, all gearframes in original stack 74 are rearranged such that each frame stack inthe second set of frame stacks 78 includes one and only one gear frame72 from each frame stack in the original set of frame stacks 74.

[0078] After reshuffling of the frame stacks is completed, each newstack 80, in the set of new stacks 78, is placed in its reactor for thesecond round of synthesis. Similar to the first round of synthesis,there are 24 reactors, each containing a reagent, with no reagentrepeated among the second set of 24 reactors.

[0079] As shown in FIG. 7, gears 70 are then liberated from the gearframes 72 and frame stacks 80 and placed on rods 82, thereby forming acolumn of gears 84. Each of frame stacks 80 yields 48 columns of gears84 placed on rod 82.

[0080] After a second randomization step illustrated in FIG. 7 column ofgears 84 are arranged into a group of gear columns 86. Each group ofgear columns 86 includes one and only one column of gears from each ofthe twenty-four set, once-reshuffled frame stacks 80. Thisre-arrangement results in 24 new groups, each group consisting of 48gear columns. The liberation of columns of gears may be done manuallyusing rods 82 that have one end having a stop-end 90. The other rod endmay be inserted through the holes in each gear.

[0081] Each of the group of gear columns 86 are then reacted with athird reagent in a third and final round of synthesis. The methodrepeats the previous synthesis steps i.e. each group of the newly formedset of twenty-four groups is placed into its own reactor, wherein noneof the twenty-four reactors has the same reagent.

[0082] After the third round of synthesis has been completed, the gearsare stored on their respective rods 82 or removed from their rods andplaced in a single layer X-Y plate for future processing, such ascleavage and extraction. One can determine the chemical composition ofthe compounds on each gear by the 2D spatial address of the gear.Because more than one plate is required to store the entire library ofcompounds in this example, a label must provide a third component toprovide a 3-D spatial identification.

[0083] In a preferred embodiment of the apparatuses, Chiron lanterns orgears or other similar supports are placed into holes in frames and heldin place by friction fit or interlocking. The means for removing thesupports from the frames can be provided in a number of ways dependingon whether the supports are attached to the frames by friction fit orwhether the supports are physically attached. If the supports are in theframes by friction fit or interlocking, the supports must be taken outfrom the frames, while preserving the spatial relationship of thesupports relative to the other supports. If the supports are physicallyattached to the frames, the supports must first be cut and thenliberated from the frames.

[0084] The gears may be pushed or pulled out from the holes of theframes using a variety of tools. One such tool already discussed is arod 82 having a stop-end 90 as shown in FIG. 7. The support, whether agear, lantern or another shape, is designed with a hole through themiddle. The rod is placed through a vertical line of support holes usingone rod end. The stop-end of the rod cannot go through the small hole ofthe supports and thus a vertical column of supports is caught on the rodand can be liberated from the frames by pulling the tip of the rod. Thesupports may be conveniently stored on the rods or the supports may belabeled and stored for later cleaving of each unique compound from thesupports.

[0085] If the supports are attached to the frame, the supports mustfirst be cut from the frame before removal. There are many conceivablevariations for liberating the supports from the frames in the last stepdependent on the specific design of the frames and supports. Some havebeen described in co-pending application Ser. No. 09/082,038. (See BaumApplication at FIGS. 25-28 for various embodiments for removing thesupports from the wells and the accompanying discussions pp. 35-40.)

[0086] Gears 70, like lanterns 10, are made of polypropylene with a thinlayer of polystyrene on its surface that has been functionalized toreact with reagents used in synthesizing the compound libraries. Asshown in FIG. 8 gear 70 comprises a tubular structure with 10 shortoutwardly projecting fins evenly spaced around the circumference of gear70. The outer diameter of gear 70 is approximately 4.0 mm to 6.0 mm,preferably around 5.0 mm. Library subunits are synthesized on allsurfaces of the gears 70.

[0087] One type of gear frame suitable for use with gears 70 is shown inFIG. 9. Gear frame 92 is made of high density polyethylene orpolypropylene. Gear frame 92 includes 96 apertures arranged in a 8×12array. The apertures extend through the thickness of gear frame 92 suchthat rods may be passed through both gears 70 and gear frame 92. Ofcourse, gear frames with greater than or less than 96 apertures may bemanufactured. Gears 70 are maintained in the apertures of gear frame 92by a friction fit or interlocking. Gear frame 92 also may be fitted withone or more radio-frequency identification (RFID) chips to confirm theidentification of gear frame 92 and gears 70 within gear frame 92.

[0088] In yet another illustrative example, manipulation of solid-phasesupports is minimized through use of a plate that functions both as acontainer to maintain the solid-phase supports in a 3D array and as areactor for the various reaction steps. In this example, a combinatoriallibrary having 576 compounds is synthesized on solid-phase supports,which comprise tubes cut into individual solid-phase support rings. Asdiscussed above and shown in FIG. 10, tube rings are structurallysimilar to lanterns, having a tubular structure with an outer diameterof approximately 7.8 mm, an inner bore diameter of approximately 6.9 mm,and a height of approximately 3.1 mm. Each tube ring supportsapproximately 15 μmols of compound, which is approximately 6 mg ofcompound at an average molecular weight of 400.

[0089]FIG. 11 illustrates a 96 wells or holes, 3-D containment plate 200which can be used in the first embodiment of this present invention.Typically such a plate will have 96 holes or more. Note thedouble-drilled first hole 130 and second hole 140. The two holesintersect and connect the two holes. The first hole is intended to be achannel or well for which the solid supports may be inserted and stackedinside. The second hole is intended to provide a separate channel forreagents to flow through and contact each stacked solid support in thefirst channel. Since the holes intersect, there is an opening betweenthe first and second channels where the reagent may pass through. Later,when the solid supports need to be retrieved, a rod having a stop-end atone end may be used to pull the stacked column of supports out of the3-D array by inserting a first end of the rod into the second channeland a bend in second end of the rod is used to catch the end of thecolumn of supports stacked in the first channel. The first end of therod can be pulled to free the friction fitted or interlocked gears fromthe frames.

[0090] C. “Sort and Combine” Synthesis Using Frames

[0091] The third embodiment, in accordance with the present invention,comprises a “sort and combine” synthesis using 2-D frames having N rowby M column of solid supports. This method is suitable for large scaleproduction of combinatorial libraries wherein the numbers of uniquecompounds exceed 100,000. A frame is prepared by placing supports havingthe same monomer or building block into the first column, filling all Nplaces. The second column of the frame is filled with another set ofsupports all having the same monomer but different from the monomer inthe first column. Each column is thus filled with supports havingdifferent monomers attached to the supports.

[0092] If Q numbers of identical frames are used, prepared as describedabove, there should be Q reactors, each having a different reagent. Eachframe numbered 1 through Q is immersed in its own reactor to allow thesupports on the frames to react with a reagent. After this step, eachframe 1 through Q is then taken from the reactors and physically splitinto subframes of rows of the original frame. Next, all of the subframesare reassembled in groups such that all of the same numbered rows 1 ofeach original N×M frame are assembled into one group of subframes, allrows 2 of each original frame are assembled into another group ofsubframes and so on until the last, Nth row of each original N×M frameis assembled into a group of subframes. After reassembly there are Ngroups of subframes. In the second synthesis step, each of these groupsin turn is immersed into N number of different reagents to provide M×N×Qdiversity. Q, which represents the number of original frames and alsothe number of reactors, is independently chosen.

[0093]FIG. 12 provides an example of the preferred implementation of themethod in accordance with the present invention. While the method may beused to synthesize highly complex libraries, i.e., greater than about100,000 compounds per library, the following example illustratessynthesis on a much smaller scale in order to provide a simplified, yetcomplete, explanation of the method.

[0094] In this example, a library of only 27 different compounds will besynthesized on solid-phase supports. Each final compounds is composedonly of three subunits or building blocks: A, B, and C. The 27 compoundsare ultimately arranged in a 2D spatial array, wherein the chemicalcomposition of the compound may be determined by its unique 2D spatialaddress.

[0095] Many known types of free, solid supports may be used with thismethod. We have already described Chiron lanterns and gears which may befriction-fitted into the holes in frames. We will assume in this examplethat Chiron lanterns 10, as depicted in FIG. 13, are used.

[0096] Referring to FIG. 12, in the first round of synthesis, 27identical lanterns 10 are reacted with a first reagent A, B, or C, in amanner known in the art, e.g., as described in the Lebl '696 patent. The27 solid-phase supports 10 are evenly distributed into three reactionflasks 20 a, 20 b, and 20 c. The flasks are essentially reactors exceptthat flasks have smaller volumes. After the first synthesis step iscompleted, nine lanterns in flask 20 a will have attached the A subunit,nine lanterns in flask 20 b will have attached the B subunit, and ninelanterns in flasks 20 c will have attached the C subunit.

[0097] The groups of nine lanterns from each flask are then rearrangedinto the holes of three lantern frames 30 a, 30 b, and 30 c byfriction-fitting the lanterns. It is necessary that each lantern framebe provided equal numbers of lanterns from each flask in an orderlyarrangement. In this case each flask A, B and C contributes threelanterns. Note that in this example, each frame 30 a, 30 b and 30 c hasthe identical 2-D spatial arrangement of lanterns. For each lanternframe 30 a-c, lanterns from first flask 20 a are placed in the firstcolumn (c1), lanterns from second flask 20 b are placed in column (c2),and lanterns from third flask 20 c are placed in the column (c3)farthest to the right. Note that this is but one example of a workableorderly arrangement. Other arrangements can serve equally well as longas each frame is provided lanterns in equal numbers from each differentflask available, and the arrangement is orderly and known.

[0098] In the second synthesis step, each frame 30 a, 30 b, and 30 c isthen reacted with a second set of reagents, also having subunits A, B,and C, by immersing each frame into its respective reactors, 40 a, 40 band 40 c. Note that subunits may be the same subunits in the firstsynthesis step as the example provided. However, within a synthesisstep, each subunit provided in each reactor should be unique.

[0099] As a result of the second round of synthesis, the lantern frames30 a, 30 b, 30 c contain nine different two-subunit synthesisintermediates. The lanterns in frame 30 a will have three differentsynthesis intermediates as follows: in column c1, three lanterns havingthe intermediates AA; in column c2, three lanterns having theintermediates AB; and in column c3, three lanterns having theintermediates AC. The lanterns in frame 30 b will have three differentsynthesis intermediates as follows: in column c1, three lanterns havingthe intermediates BA; in column c2, three lanterns having theintermediates BB; and in column c3, three lanterns having theintermediates BC. Similarly, the lanterns in frame 30 c will have threedifferent synthesis intermediates as follows: in column c1, threelanterns having the intermediates CA; in column c2, three lanternshaving the intermediates CB; and in column c3, three lanterns having theintermediates CC.

[0100] The next step provides a randomization. Each row of the frame 30a is then broken into smaller subframes of rows as indicated bysubframes 50 a, 50 b, and 50 c. Each split subframe has three supports.Frame 30 b is broken into smaller subframes 52 a, 52 b and 52 c. Andsimilarly, frame 30 c is broken into subframes 54 a, 54 b and 54 c. Thesubframes are regrouped such that all split frames from the same rowsare grouped together. For example, subframes from the first rows, 50 a,52 a and 54 a are grouped into new group of subframes 60 a. Subframesfrom the second rows, 50 b, 52 b and 54 b are grouped into a new groupof subframes 60 b. Similarly, subframes from the third rows, 50 c, 52 c,and 54 c are grouped into new group of subframes 60 c.

[0101] In the third synthesis step, each new group of subframes, 60 a,60 b and 60 c, is immersed into reactors 40 a, 40 b, and 40 c,respectively. Note that in this example, the same reactors that wereused in the second round of synthesis are used again in this third roundof synthesis. Alternatively, other reactors (not shown) having differentsubunits, e.g., H, I, and J may be used. After the third round ofsynthesis, all 27 of the possible three unit combinations of buildingblocks A, B, and C will have been synthesized. Each compound will beattached to and located on one and only one lantern.

[0102] In an alternative embodiment, smaller frames may be used in lieuof a breakable larger frame. Specifically, in this example, rather thanusing the three lantern frames 30 a-c, which are adapted to contain 9lanterns apiece, one can use nine smaller frames, which are adapted tocontain 3 lanterns apiece.

[0103] All 27 lanterns 10 are then removed from their respectivesubframes, and transferred to plate 65 comprising a 3 row by 9 column(3×9) array of wells. The compounds are then removed from the lanterns,such that there is one unique compound per well. Therefore, eachcompound has a unique location or spatial 2D address within the plate,i.e., row (1-3), column (1-9), and may therefore be identified by itsunique spatial 2D address. For example, compounds located in well at r2,c5 will have a chemical composition comprising BBB. According to thespatial 2D address, one can therefore determine the chemical compositionof the compound.

[0104] As explained above, each lantern 10 moves through a given patternthroughout synthesis such that its ultimate location or spatial addressreveals the chemical composition of the compound attached to eachlantern. In addition, the spatial address of each compound and itsassociated chemical composition will also reveal the history of thesynthesis, including the various rounds or reactions of synthesis. Forexample, by its spatial address within the 3×9 plate, it may bedetermined which flask the compound originated from in the first roundof synthesis. The spatial address therefore contains a wealth of usefulinformation about the compound.

[0105] In addition, the present method is not limited to any particularpattern or grouping of solid-phase supports. Any ordered, nonrandompattern or grouping may be incorporated into the present method as longas the relationship between the pattern and the ultimate spatial addressof the library compounds is determinable. For example, in an alternativeembodiment of the present method, the lanterns may first be arrangedsuch that the 9 lanterns from the first flask comprise the first row(rather than column) of each 3×3 lantern frame.

[0106] Lantern 10 is known in the art and is commercially available fromChiron. Lantern 10 is made of polypropylene with a thin layer ofpolystyrene on its surface similar to other solid-phase supports knownin the art. This polystyrene surface is functionalized to react withreagents used in synthesizing the compound libraries. As shown in FIG.13, a lantern comprises four tubular substructures, 121, 122, 123, and124, that are attached to each other, creating an overall tubularstructure. Lantern 10 has an outer diameter of approximately 5.0 mm toapproximately 6.0 mm, preferably around 5.0 mm, and an inner bore with adiameter of approximately 2.0 mm to approximately 3.0 mm, preferablyaround 2.5 mm. In addition, the height of each lantern is approximately5 mm. In addition, each lantern 10 supports approximately 15 μmols ofcompound. Library subunits are synthesized on all surfaces of thelanterns, including both the outer and inner surface. Although lanternsare used in the preferred embodiment of the present method, it will beappreciated by those of ordinary skill in the art that any physicallymanipulable solid-phase support may be incorporated into the presentmethod. Tubes cut into individual solid-phase support rings may also beused.

[0107] A preferred type of lantern frame that may be used in the presentmethod is shown in FIGS. 14a and 14 b. Frame 32 comprises a high densitypolyethylene, polypropylene, or other chemically resistant material andhas dimensions of approximately 18 mm by 81 mm. Frame 32 includes 16wells 33 arranged in a 2×8 array, and wells 33 are dimensioned tocontain lanterns 10. In addition, frame 32 includes knife cut grooves(not shown) that allow it to be divided or broken apart into subframeshaving at least two wells apiece. Optionally, frame 32 may be fittedwith one radio-frequency identification (RFID) chips 34 known in the artto record the identity of frame 32 and lanterns 10 within frame 32. RFIDchip 34 is approximately 11 mm long and is positioned at one end offrame 32. In addition, a variety of alternative structural supports maybe incorporated into the present invention.

[0108] In sum, the apparatus comprises: (a) single frames which can bebroken into subframes; (b) reactors; and (c) means for holding solidsupports on the frames temporarily or means to allow cutting of thesupports.

[0109] The present invention may be embodied in other forms withoutdeparting from its spirit or essential characteristics. The describedembodiments are to be considered only as illustrative and not asrestrictive. For example, in each of the examples described above, eachsynthesis comprises three rounds of reactions. However, depending on thecombinatorial library desired, one may need fewer than three rounds orless than three rounds. The scope of the invention is, therefore,indicated by the appended claims.

What is claimed:
 1. A system for synthesizing a combinatorial librarycomprising: a containment device with a plurality of channels, eachchannel fluidly connected to the outside of the containment devicethrough an opening; free solid supports, wherein the solid supports aresized to stack inside the plurality of channels and thereby create acolumns of supports, wherein the spatial arrangement of the solidsupports in channels defines a 3-D array; and means for removing thesolid supports through said channel, retaining the spatial relationshipof the supports with each other.
 2. The system as in claim 1 wherein thesolid supports have a hole in the middle and the means for removing thesolid supports through the channel is a rod with one stop-end largerthan the hole in the support, said rod used to insert through the holesof a vertical stack of supports.
 3. The system as in claim 1 wherein thesolid supports are gears, having a tubular structure with a plurality ofoutwardly projecting fins.
 4. The system of claim 3 wherein the gear ismade of polypropylene with a surface of polystyrene and having an outerdiameter of from about 4.0 mm to about 6.0 mm.
 5. The system of claim 4wherein the outer diameter of each gear is about 5.0 mm.
 6. The systemas in claim 1 wherein the solid supports are rings, having an innerborehole and an outer diameter.
 7. The system of claim 6, wherein therings are made of polypropylene with a surface of polystyrene, with anouter diameter of from about 7.8 mm, an inner bore diameter of about 6.9mm, and a height of about 3.1 mm.
 8. The system as in claim 1 whereinthe solid support are lanterns, having holes in the middle.
 9. Thesystem of claim 8 wherein the lanterns are made of polypropylene with asurface of polystyrene and having a tubular structure with an outerdiameter of from about 4.0 mm to about 6.0 mm and an inner bore diameterof from about 2.0 mm to about 3.0 mm.
 10. The system of claim 9 whereinthe outer diameter of each lantern is about 5.0 mm, and the inner borediameter is about 2.5 mm.
 11. The system as in claim 1 wherein the meansfor removing the solid supports through the channel is a vacuum thatpicks up X-Y layers of solid-phase supports simultaneously.
 12. Thesystem as in claim 1 wherein the channels are cylindrical wells.
 13. Thesystem as in claim 1 wherein the solid supports stacked in one channelhave mechanical means of linking supports, and the means of removal ofthe entire stack of supports in one channel is to grasp the firstsupport closest to the channel opening, thereby retaining the spatialrelationship of the supports to the other removed supports.